Metabolism and antioxidant activity of SlGSTD1 in Spodoptera litura as a detoxification enzyme to pyrethroids

Glutathione S-transferase (GSTs) are members of multifunction enzymes in organisms and mostly known for their roles in insecticide resistance by conjugation. Spodoptera litura (Fabricius) is a voracious agricultural pest widely distributed in the world with high resistance to various insecticides. The function of GSTs in the delta group of S. litura is still lacking. Significantly up-regulation of SlGSTd1 was reported in four pyrethroids-resistant populations and a chlorpyrifos-selected population. To further explore its role in pyrethroids and organophosphates resistance, the metabolism and peroxidase activity of SlGSTD1 were studied by heterologous expression, RNAi, and disk diffusion assay. The results showed that Km and Vmax for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity of SlGSTD1were 1.68 ± 0.11 mmol L−1 and 76.0 ± 2.7 nmol mg−1 min−1, respectively. Cyhalothrin, beta-cypermethrin, and chlorpyrifos had an obvious inhibitory effect on SlGSTD1 activity, especially for fenvalerate, when using CDNB as substrate. Fenvalerate and cyhalothrin can be metabolized by SlGSTD1 in E. coli and in vitro. Also, silencing of SlGSTd1 significantly increased the toxicity of fenvalerate and cyhalothrin, but had no significant effect on the mortality of larvae treated by beta-cypermethrin or chlorpyrifos. SlGSTD1 possesses peroxidase activity using cumene hydroperoxide as a stress inducer. The comprehensive results indicate that SlGSTD1 is involved in fenvalerate and cyhalothrin resistance of S. litura by detoxication and antioxidant capacity.

Our previous study has indicated that SlGSTd1, a GSTs gene from delta cluster, is significantly overexpressed in four field-collected populations (LF, NJ, JD and CZ) resistant to pyrethroids 21 . Zhang et al. 19 also reported the significantly higher expression level of SlGSTd1 in a chlorpyrifos-selected strain than the susceptible strain. Does the overexpression of SlGSTd1 relate with pyrethroids and chlorpyrifos resistance? Heterologously expression and RNAi were adopted to investigate the contribution of SlGSTd1 in insecticides resistance. The findings enriched the knowledgement of GSTs gene from delta cluster and also revealed the insecticides resistance mechanism in S. litura.

Results
The expression of SlGSTD1 in E. coli and kinetic properties. To evaluate the role of SlGSTd1 in pyrethroids and chlorpyrifos resistance, SlGSTd1 was heterologously expressed in Escherichia coli. Its recombinant protein was characterized by SDS-PAGE and kinetic properties were determined using 2,4-dinitrochlorobenzene (CDNB) as a standard substrate. The theoretical molecular mass of SlGSTD1 is predicted to be 24.3 kDa by Compute pI/Mw (https:// web. expasy. org/ compu te_ pi/), conforming with the indicated band in lane 3 and lane 4 marked with red box in Fig. 1A (between 20 and 26 kDa). The recombinant protein SlGSTD1 showed high catalysis activity to CDNB, and its K m and V max values were 1.68 ± 0.11 mmol L −1 and 76.0 ± 2.7 nmol mg −1 min −1 , respectively (Fig. 1B).

Inhibition of insecticides on the activity of SlGSTD1.
To determine the competition binding ability of fenvalerate, beta-cypermethrin, cyhalothrin and chlorpyrifos to SlGSTD1 conjugating activity against CDNB, a set of experiments was carried out using diethyl maleate (DEM, the inhibitor of GSTs) as a positive control. As shown in Fig. 2 In vitro metabolism activity of SlGSTD1 to insecticides. In order to determine the metabolic activity of purified recombinant protein SlGSTD1 to insecticides, the residues of insecticide in the mixture after incubation were detected by ultrahigh-performance liquid chromatography (UPLC). As shown in Table 1, the residual peak area of fenvalerate and cyhalothrin incubated with SlGSTD1 for 3 h decreased significantly compared with that incubated with potassium buffer saline (PBS) or boiled SlGSTD1. While the residual peak area of alphacypermethrin, theta-cypermethrin and chlorpyrifos incubated with SlGSTD1 had no significant reduction compared with PBS or boiled SlGSTD1. Silencing of SlGSTd1 increased the susceptibility of S. litura to fenvalerate and cyhalothrin. To validate the involvement of SlGSTd1 in insecticides resistance by in vivo data, RNAi of SlGSTd1 was accomplished by feeding larvae with artificial diet containing dsRNA. As shown in Fig. 4A, the relative expression level of SlGSTd1 decreased significantly after dsSlGSTd1 feeding for 12 and 24 h, when compared to the treatment of dsGFP and H 2 O, respectively. In addition, compared with dsGFP and H 2 O, the relative expression level of SlGSTd1 showed no significant change after feeding on dsSlGSTd1 for 48 h. Bioassay results showed that silencing of SlGSTd1 significantly increased the cumulative mortality after fenvalerate treated for 72 h (Fig. 4B) and cyhalothrin treated for 48, 60 and 72 h (Fig. 4C). The cumulative mortality after treatment with beta-cypermethrin and chlorpyrifos had no significant change after feeding on dsGFP or dsSlGSTd1 (Fig. 4D,E).
Antioxidant activity of SlGSTD1 in E. coli against CHP. To further characterize the antioxidant activity of SlGSTD1, the halo diameter of inhibition zones in LB plates spread with E. coli expressing pET-26b(+)/SlGSTd1 or pET-26b(+) was measured using CHP as a stress inducer. When the concentrations of CHP were 50, 100, 200, and 300 mmol L −1 , the inhibition zone halo diameter of E. coli cells expressing pET-26b(+)/SlGSTd1 was significantly lower than that of E. coli cells expressing pET-26b(+), with inhibition rates of 32.0%, 15.5%, 33.9% and 19.5%, respectively (Fig. 5A,B).    The relative expression of SlGSTd1 in S. litura after larvae feeding on dsRNA or H 2 O. Different lowercase letters indicated significant differences analyzed by ANOVA followed by Tukey's HSD test (P < 0.05). (B-E) Mortality of larvae treated by fenvalerate (B), cyhalothrin (C), beta-cypermethrin (D), chlorpyrifos (E) after feeding on dsRNA. * Indicated significant differences in larvae mortality between feeding on dsSlGSTd1 and dsGFP (student's t-test, P < 0.05).

Discussion
As one of the three major detoxification enzymes in the organism, GSTs have been well demonstrated to be involved in the detoxification of both endogenous and xenobiotic compounds, such as insecticides 31 and plant allelochemicals 32 . In this study, SlGSTD1 in S. litura was demonstrated to play roles in fenvalerate and cyhalothrin resistance by metabolism activity or its antioxidant activity. Cytosolic GSTs are hetero-or homo-dimeric proteins with a molecular weight around 25 kDa 7 . Here, the molecular weight of SlGSTD1 is identified around 26 kDa by SDS-PAGE electrophoresis, similar to its theoretical value. The kinetic properties of recombinant protein SlGSTD1 were determined using CDNB as a substrate. The results showed that the K m and V max values of SlGSTD1 were 1.68 ± 0.11 mmol L −1 and 76.0 ± 2.7 nmol min −1 mg −1 (Fig. 1B), indicating that SlGSTD1 was successfully expressed in E. coli. The IC 50 value of insecticides on GST activity inhibition can represent the affinity of insecticide to GST enzyme, and this affinity is related to the metabolic ability of insecticide 15,33 . As shown in Fig. 2, the IC 50 value of fenvalerate was lower than that of cyhalothrin, beta-cypermethrin and chlorpyrifos, indicating that fenvalerate had a higher affinity for competitive binding to SlGSTD1. The significant overexpression of GSTs genes in insecticides-resistant populations is often deduced to play role in insecticides resistance 34 . However, the relationship still needs further validation, and metabolism activity could provide the most direct evidence of gene function. SIGSTd1 is highly likely to be involved in the detoxifying of pyrethroids and chlorpyrifos for its significant overexpression in pyrethroid-resistant populations 21 and a chlorpyrifos-selected strain 19 . Studies have suggested that pyrethroids could be metabolized by insect GSTs. For example, HaGST-8 in Helicoverpa armigera could effectively metabolize cypermethrin in an aqueous solution 35 . CpGSTd1, CpGSTd3 and CpGSTe3 in Cydia pomonella could metabolize lambda-cyhalothrin, a most commonly used insecticide for C. pomonella control [14][15][16] . In this study, a delta GST in S. litura, SlGSTD1, was found to have metabolism activity to fenvalerate and cyhalothrin in E. coli and in vitro. Additionally, the cumulative mortality of larvae applied by fenvalerate and cyhalothrin was increased significantly after the silencing of SlGSTd1. However, SlGSTD1 could not metabolize beta-cypermethrin or chlorpyrifos directly either in vitro or in E. coli. Our previous study also showed that beta-cypermethrin could not be metabolized directly by SlGSTE9, SlGSTE12 or SlGSTO2, either 24,25,30 . But these GSTs might play a role in beta-cypermethrin resistance by sequestration or its antioxidant activity, which needs further study. Although SlGSTd1, SlGSTe9, SlGSTe12 and SlGSTo2 were both overexpressed in pyrethroid-resistant populations, their recombinant proteins have different metabolism spectrum for pyrethroids 24,25,30 . These findings enriched the growing body of evidence that the GSTs can deal with a variety of xenobiotic compounds through substrate diversity and specificity.
In some insects, the GST isoenzyme, acting as an independent peroxidase, has been thought to aid in acellular antioxidant defense by reducing organic hydroperoxides within membranes and lipoproteins 36 . Vontas et al. found that the resistance of Nilaparvata lugens to permethrin was caused by the peroxidase activity of GSTs, and concluded that GSTs were involved in the resistance to pyrethroids by protecting insect tissues from peroxidation damage 37 . GSTD1, GSTD2, GSTD3, GSTD7, GSTD9 and GSTD10 in Drosophila melanogaster were found to have 4-hydroxynonenal conjugating activity, indicating their potential to reduce oxidative stress 38 . Notably, only GSTD1 (expressed as DmGSTD1-1) showed glutathione peroxidase activity against substrate CHP 38 . In Culex pipiens, CpGSTD1 exhibited peroxidase activity with CHP, while CpGSTD2 showed no such activity 39 . Our study showed that SlGSTD1 had peroxidase activity, similar to SlGSTE9, SlGSTE12 and SlGSTO2. Based on the above findings, it is deduced that GSTs may play a role in the antioxidant defense of cells against pesticide induced oxidative damage, thereby contributing to insecticides resistance.
In conclusion, SlGSTD1 in S. litura can metabolize fenvalerate and cyhalothrin both in E. coli and in vitro. Fenvalerate had a strong affinity to SlGSTD1. The silencing of SlGSTd1 significantly increased the toxicity of

Materials and methods
Insect culture. A population of S. litura (NJ) originally collected from Nanjing, Jiangsu province, China, is used in this study. NJ population had high level of resistance to pyrethroids, and low or no resistance to phoxim, profenofos, chlorpyrifos, emamectin benzoate, chlorantraniliprole, cyantraniliprole, imidacloprid, or methomyl. Its rear condition is the same as the description in Xu et al. 21 . Briefly, larvae were fed with artificial feed under the conditions of 27 ± 1 °C, 70% relative humidity, and a photoperiod of 12 h light and 12 h dark until pupal stage. Adults were provided with 10% honey solution.
Expression and purification of SlGSTD1. Total RNA was extracted from the third instar larvae. The first-strand cDNA was synthesized from 1 μg RNA according to the instructions of FastQuant RT Kit (Tiangen, Beijing, China). The coding sequence of SlGSTd1 was amplified by PCR with primer pairs added with NdeI and XhoI ( Table 2). The PCR products were inserted to pET-26b(+) and the expression vector pET-26b(+)/SlGSTd1 was constructed. The constructed expression vector was then transformed into E. coli BL21 ( . After the addition of CDNB (50 μL, 50 μmol mL −1 ), the mixture was shaken quickly and A 340 Table 2. Primer pairs used in this study. Nucleotides bold were restriction enzyme sites. Nucleotides underlined were T7 RNA polymerase promoter sequence.

SlGSTd1F
CAT ATG GCT TTA GCT CTA TAC TAC  cDNA full length clone  SlGSTd1R  CTC GAG CAA CTC AGT TTT TGC TTT GA   dsSlGSTd1-F1  GGA TCC TAA TAC GAC TCA CTA TAG GGCA GCC CTT AAC ATCCA   dsRNA clone of SlGSTd1  dsSlGSTd1-R1  CGG TAG CGT CAA TCG TAG   dsSlGSTd1-F2  GCA GCC CTT AAC ATCCA   dsSlGSTd1-R2  GGA TCC TAA TAC GAC TCA CTA TAG GCGG TAG CGT CAA TCG TAG   dsGFP-F1  GGA TCC TAA TAC GAC TCA CTA TAG GCAC CCT CGT GAC CAC CCT G   dsRNA clone of GFP  dsGFP-R1  TTG ATG CCG TTC TTC TGC TTG   dsGFP-F2  CAC CCT CGT GAC CAC CCT G   dsGFP-R2  GGA TCC TAA TAC GAC TCA CTA TAG GTTG ATG CCG TTC TTC TGC TTG   SlGSTd1qF  TAA GTT GAC CTT GGC TGA CC  qRT-PCR  SlGSTd1qR TAT Metabolism activity analysis to insecticide. The metabolism activity of SlGSTD1 towards fenvalerate, beta-cypermethrin, cyhalothrin and chlorpyrifos was determined by UPLC. The procedure was the same as our previous study 24 . For in vitro assay, the purified recombinant protein (180 μL, 1 mg mL −1 ) was mixed with PBS (250 μL, 0.1 mol L −1 , pH 6.8) and preheated at 37 °C, then added with insecticide (20 μL, 500 mg L −1 ). Finally, freshly prepared GSH (50 μL, 100 μmol mL −1 ) was added to start the reaction. The reaction system was immediately placed in a water bath and incubated at 37 °C for 3 h. The reaction was terminated by adding the same volume of acetonitrile and saturated with sodium chloride to extract the insecticide. After shaken at 200 rpm for 2 h and brief centrifugation at 3000 rpm for 2 min, the supernatant was carefully absorbed and filtered through a 0.22 μm membrane. The extracts were detected by Waters Acquity UPLC system with an Acquity UPLC BEH C18 analytical column (2.1 mm × 100 mm, 1.7 μm). The chromatographic conditions were acetonitrile/water = 80/20 (v/v), flow rate at 0.4 mL min −1 and 5 μL injection volume. Fenvalerate, beta-cypermethrin, cyhalothrin, and chlorpyrifos were detected and quantified at 220, 220, 230 and 290 nm, respectively. As a chiral insecticide, beta-cypermethrin could be separated into alpha-cypermethrin and theta-cypermethrin under these detection conditions. The retention time for fenvalerate, alpha-cypermethrin, theta-cypermethrin, cyhalothrin, chlorpyrifos were 1.86, 1.76, 1.67, 1.69, 1.36 min, respectively. PBS or boiled purified recombinant protein was used as control.
For in E. coli assay, the concentration of pET-26b(+)/SlGSTd1 transformed E. coli in LB liquid medium was diluted to OD 600 = 1. A total of 2 mL of the E. coli culture was added to a 50 mL LB liquid medium, containing kanamycin (50 mg L −1 ) and insecticide (50 mg L −1 for chlorpyrifos or 25 mg L −1 for fenvalerate, beta-cypermethrin, cyhalothrin). The mixture was further cultured at 37 °C, 120 rpm for 6 h, and then IPTG (1 mmol L −1 ) was added to induce SlGSTD1 expression. The mixture (2 mL) was sampled after cultured for 0, 24, 48, 72 and 96 h. Each treatment was performed in triplicate. The insecticides extraction and detection conditions were the same as the above methods. The control group was treated with LB liquid medium or E. coli transformed with pET-26b(+).

Silencing of SlGSTd1 by RNAi. Silencing of SlGSTd1 was conducted with T7 RiboMAX™ Express RNAi
System (Promega, Madison, WI). Briefly, DNA fragments of SlGSTd1 appended to a T7 polymerase promoter were amplified with primer pairs dsSlGSTd1F-1, dsSlGSTd1R-1 and dsSlGSTd1F-2, dsSlGSTd1R-2 ( Table 2). The products were purified and used for dsRNA synthesis. dsRNA of green fluorescent protein (GFP) was also obtained by the methods above and the concentration of dsRNA was determined by NanoDrop 2000 (Thermo Scientific, Foster, CA, USA).
The third instar larvae of NJ population were fed artificial diet containing 3 μg dsRNA after starved for 6 h. The artificial diet containing ddH 2 O or dsGFP under the same conditions was used as control. Quantitative real-time PCR (qRT-PCR) was conducted to determine the dsRNA interference effective after 12, 24, 48 h treatment. The qRT-PCR tests were performed with SuperReal PreMix Plus (SYBR Green, Tiangen, Beijing, China) using a QuantStudio 6 Real-Time PCR System (Applied Biosystems by Life Technologies, Foster, CA, USA). The PCR mixture (20 μL) contained 10 μL of 2× SuperReal PreMix solution, 0.4 μL of 50× ROX reference dye, 1 μL of the cDNA template, 0.6 μL of each primer, and 7.4 μL ddH 2 O. The thermal cycling was set as: 94 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. EF1α and RPL10 were used as reference genes to normalize the expression of SlGSTd1 40 . Bioassay was performed by applying 1 μL insecticide at LD 20 to the thoracic dorsum of larvae by Hamilton syringe after 12 h of treatment with dsRNA or ddH 2 O. Each insecticide treatment consisted of 12 larvae and repeated 3 times. The mortality was checked after insecticides treated for 12, 24, 36, 48, 60, 72 h. Antioxidant activity assay. A disc diffusion assay was conducted in reference to Labade et al. 35 with slight modification to determine the antioxidant activity of SlGSTD1. The LB liquid medium containing E. coli transformed with pET-26b(+)/SlGSTd1 (OD 600 = 1.0) was distributed on LB agar plates (50 mg L −1 for kanamycin, 1 mmol L −1 for IPTG) and incubated at 37 °C for 1 h. The E. coli solution containing pET-26b(+) was used as control. The filter papers (5 mm diameter) were immersed in CHP (J&K, Beijing, China) at 0, 50, 100, 200, 300 mmol L −1 dissolved in acetone. All filter papers were placed on the surface of LB agar plates and incubated at 37 °C for 36 h. The diameter of the bacteriostatic zone around the disk was measured. Each concentration was repeated 3 times and the experiment was performed twice.
Statistical analysis. The qRT-PCR results were calculated according to the 2 −△△Ct method and expressed as means ± standard deviation (SD). The student's t-test was used to analyze the statistical differences in the cumulative mortality of third instar larvae, and the disc diffusion assay. One-way ANOVA followed by Tukey's HSD test was used to analyze the statistical difference of the metabolism activity of SlGSTD1 in vitro, the expression of SlGSTd1 mediated by RNAi and the metabolism activity of SlGSTD1 in E. coli. SPSS 16.0 (IBM, Chicago, IL, U.S.A., URL:https:// www. ibm. com/ search? lang= en& cc= us&q= SPSS) was used for statistical analysis and the P values less than 0.05 were considered statistically significant.

Data availability
All data generated or analyzed during this study are included in this published article.